Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1alpha was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between -418 and -223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between -300 and -251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at -271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1alpha but instead requires the activation of PI3K/Rho/ROCK and c-Myc.     

缺氧诱导因子与肾细胞癌关系的研究进展

缺氧诱导因子(hypoxia inducible factor,HIF)对维持肿瘤细胞的能量代谢、肿瘤血管生成、促进肿瘤细胞增殖和转移起着重要作用,是肿瘤细胞低氧条件下产生的关键信号分子。本综述旨在总结前人研究,阐述HIF与肾癌细胞之间的内在关系。HIF成员是参与肾癌细胞对缺氧应答反应中的关键因子,并通过靶基因的调节,促进新生血管的生成,导致肿瘤生长。其中,HIF-1α及HIF-2α在促进新生血管的生成方面发挥着主要作用。HIF-1α及HIF-2α与VEGF密切相关,随着其的表达增高,VEGF在数量上及m RNA水平上均显著增高,显示其可通过调控VEGF参与肾癌血管生成,而HIF-2α转录激活VEGF m RNA的特异性较HIF-1α更强。HIF-3α可能存在的负性调控作用,其异构体-4的作用可能与HIF-lα的负性调节有关,其可以阻止HIF-lα与下游靶基因的缺氧反应元件(hypoxia response elements,HRE)结合,同时可在转录水平抑制HIF-lα。HIF在未来可能有成为肾细胞癌治疗的靶点。     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O(2)) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O(2), 5 h) using the matrigel assay. To further examine the role of HIF-1alpha in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1alpha (DNHIF-1alpha). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1alpha. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression

We have previously demonstrated the roles of RhoA, Rac1, and Cdc42 in hypoxia-driven angiogenesis. However, the role of oncogenes in hypoxia signaling is poorly understood. Given the importance of Rho proteins in the hypoxic response, we hypothesized that Rho family members could act as mediators of hypoxic signal transduction. We investigated the cross-talk between hypoxia and oncogene-driven signal transduction pathways and explored the role of Rac1 on hypoxia-induced hypoxia-inducible factor (HIF)-1α and VEGF expression. Since the phosphatidylinositol 3'-kinase (PI3K) pathway is involved in signal transduction of many oncogenes, we explored the role of PI3K on Rac1-mediated expression of HIF-1α and VEGF in hypoxia. We showed that LY-294002, a PI3K inhibitor, suppressed HIF-1α and VEGF induction under hypoxic conditions by up to 50%. Activation of Rac1 resulted in an upregulation of hypoxia-induced HIF-1α expression, which was blocked by LY-294002. These data suggested that Rac1 is an intermediate in the PI3K-mediated induction of HIF-1α. Interestingly, there was a significant downregulation of the tumor suppressor genes p53 and von Hippel-Lindau tumor suppressor (VHL) in cells expressing a constitutively active form of Rac1. Rac1-mediated inhibition of p53 and VHL could therefore be implicated in the upregulation of HIF-1α expression.     

Regulation of angiogenesis and tumor growth by p110 alpha and AKT1 via VEGF expression

Recent studies demonstrate that PI3K activation and PTEN mutation are frequently found in many human cancer cells and tissues. However, the mechanism of PI3K signaling in human cancer tumorigenesis remains to be elucidated. In this study we specifically downregulated p110alpha expression in ovarian cancer cells using siRNA interference. We found that p110alpha downregulation greatly decreased ovarian tumor growth and angiogenesis, and that p110alpha siRNA inhibited VEGF expression through decreasing hypoxia-inducible factor 1alpha expression in both ovarian cancer cells and tumor tissues. To determine the downstream targets of PI3K in regulating tumor growth and angiogenesis, we find that AKT1 is a major downstream mediator for regulating tumor growth, angiogenesis, and VEGF expression. These data show that p110alpha and AKT1 play an important role in tumor growth by inducing angiogenesis and by increasing HIF-1alpha and VEGF expression. This work provides a better understanding of the molecular mechanism of human cancer induced by the activation of PI3K signaling.     

Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF-1alpha and VEGF

Retinal neovascularization (NV) occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in blindness. Vascular endothelial growth factor (VEGF) is an essential growth factor for angiogenesis, and is particularly regulated by hypoxia inducible factor-1alpha (HIF-1alpha) under hypoxic conditions. Therefore, HIF-1alpha and VEGF could provide targets for therapeutic intervention on retinal NV. In this study, we investigated the inhibitory effects of small interfering RNA (siRNA) targeting HIF-1alpha and VEGF on the expression of HIF-1alpha and VEGF in human umbilical vein endothelial cells (HUVEC) in vitro and on retinal NV in vivo. siRNA-expressing plasmids targeting human HIF-1alpha (HIF-1alpha siRNA) and human VEGF(165) (VEGF siRNA) were constructed. They were transfected and co-transfected to HUVEC and C57BL/6J mice of ischemic retinopathy model. HIF-1alpha siRNA and VEGF siRNA specifically downregulated HIF-1alpha and VEGF at both mRNA and protein levels in vitro and in vivo. Neovascular tufts and neovascular nuclei were decreased in gene therapy group compared to control hypoxia group. Co-transfection of HIF-1alpha siRNA and VEGF siRNA resulted in maximal effects on VEGF suppression in vitro and in vivo. It also manifested the maximal inhibitory effect on retinal NV. These results indicate that the application of HIF-1alpha siRNA and VEGF siRNA technology holds great potential as a novel therapeutic for retinal NV.     

Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-alpha (HIF-1alpha), but rapidly effected degradation of von Hippel-Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1alpha protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1alpha up-regulation. Thus, vHL/HIF-1alpha has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.